Effects of bFGF and PDGF-BB on osteoblast differentiation of bone marrow-derived mesenchymal stem cell in rat

ABSTRACT

In this study we evaluate the effects of bFGF-BB and PDGF on in vitro proliferation, differentiation and mineralization of mesenchymal stem cells (MSCs) from rat. MSCs were prepared from the bone marrow of 6 or 7-week-old male rats with a technique previously described by Maniatopoulos et al. in 1988. Lineage differentiation to osteogenesis, chondrogenesis and adipogenesis were performed. At first, we characterized the cultured cell on passage 1, 3, 5, 7 with immunocytochemical staining using CD29, 44, 34, 45, alpha-SMA and type I collagen. And to study the effects of bFGF and PDGF-BB on proliferation, differentiation and mineralization, we seeded the expanded cell at a density of 6 x 10(3) cells/cm2 to 100-mm dish for evaluation of cell proliferation and MTT assay was carried out on day 2, 4, 7, 9. We also resuspended the cells with same density (6 x 10(3) cells/cm2) to 24 well plates for subculture. On the following day, the attached cells were exposed to 2.5ng/ml bFGF and/or 25ng/ml PDGF-BB daily during 5 days. The osteocalcin (OC) level was assessed and mineral contents were evaluated with alizarin red S staining on subculture day 2, 7, 14, 21. We identified the mesenchymal stem cell from the bone marrow derived cells of rat through their successful multi-differentiation and stable display of its phenotype. And bFGF and PDGF-BB showed the effect that inhibited osteoblastic differentiation and mineralization mildly in above concentration at in vitro culture.